High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules

Abstract

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays. A structure–activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity.

Therapeutic Targeting of Glutaminolysis as a Novel Strategy to Combat Cancer Stem Cells

“…On the other hand, compound 968 is a pan-glutaminase inhibitor with a four-fold higher potency in inhibiting GLS2 than GLS1 [ 91 ]. Recently, many other glutaminase inhibitors, such as C9.22 [ 92 ], compound 27 (IPN60090) [ 93 ], alkyl benzoquinones [ 94 ], and thiazolidine-2,4-dione compounds [ 95 ] were developed and are undergoing evaluation.…”

“…Compound 968 is a pan-glutaminase inhibitor that interacts with both kidney-type glutaminase (KGA) and glutaminase C (GAC) isoforms of GLS1 by preventing the combination of inactive monomers of GLS1 into an active tetramer [ 92 , 103 ]. Moreover, compound 968 has an inhibitory effect on GLS2, which was previously identified as being essential in the tumorigenesis of luminal-type breast cancers [ 104 ].…”

“…Recently, a high-throughput screening study using the coupled enzyme-based fluorescent glutaminase activity assay to screen a library of about 30,000 compounds was conducted [ 92 ]. As a result, 11 glutaminase inhibitors were found to be hits, and they were further characterized by in silico, biochemical, and glutaminase-based cellular assays [ 92 ].…”

“…Recently, a high-throughput screening study using the coupled enzyme-based fluorescent glutaminase activity assay to screen a library of about 30,000 compounds was conducted [ 92 ]. As a result, 11 glutaminase inhibitors were found to be hits, and they were further characterized by in silico, biochemical, and glutaminase-based cellular assays [ 92 ]. The structure-activity relationship research on the most promising hit (C9) led to the identification of C9.22, a derivative with improved in vitro and cellular glutaminase-inhibiting activity [ 92 ].…”

“…As a result, 11 glutaminase inhibitors were found to be hits, and they were further characterized by in silico, biochemical, and glutaminase-based cellular assays [ 92 ]. The structure-activity relationship research on the most promising hit (C9) led to the identification of C9.22, a derivative with improved in vitro and cellular glutaminase-inhibiting activity [ 92 ]. C9.22 inhibited GAC selectively, presumably through a mechanism similar to BPTES and CB-839.…”